Mitotic entry and exit in most organisms is controlled by the synthesis and destruction of cyclin B, a positive regulatory subunit of the protein kinase Cdc2, the catalytic component of mitosis promoting factor (MPF) (Norbury et al. (1992) Ann. Rev. Biochem. 61:441-470; Murray (1995) Cell 81:149-152). Cyclins are marked for destruction by the covalent addition of ubiquitin at the end of mitosis (Glotzer et al. (1991) Nature 349:132-138; Hershko et al. (1991) J. Biol. Chem. 266:16376-16379; Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). Ubiquitinated cyclins are then rapidly degraded by the 26S proteasome (Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). This process is catalyzed by a cyclin-specific ubiquitin ligase, E3-C, which is part of a 20S particle, the cyclosome (Sudakin et al (1995) Mol. Biol. Cell. 6:185-198). Cyclosome activation is initiated by Cdc2 (Felix et al. (1990) Nature 346:379-382; Sudakin et al. (1995) Mol. Biol. Cell. 6:185-198) and terminated by an okadaic acid-sensitive phosphatase (Lahav-Baratz et al. (1995) Proc. Nat. Acad. Sci. USA, in press). This particle contains homologs of two yeast proteins, Cdc16 and Cdc27 (King et al. (1995) Cell 81:279-288), proteins required for the destruction of cyclin B and the metaphase-anaphase transition (Tugendreich et al. (1995) Cell 81:261-268; Irniger et al (1995) Cell 81:269-277).
Cyclosome-associated E3-C catalyzes cyclin ubiquitination using a specialized ubiquitin conjugating enzyme or carrier protein (E2); also called Ubc, originally identified in clam as E2-C (Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). Multiple species of E2's were first found in animal cells (Pickart et al (1985) J. Biol. Chem. 260:1573-1581), and at least ten different Ubc's have now been identified in yeast (Jentsch (1992) Ann. Rev. Genetics 26:179-207).
Structurally, all known E2's share a conserved domain of approximately 16 kD. This domain contains the cysteine (Cys) residue required for the formation of ubiquitin-E2 thiol ester. Certain E2 enzymes contain additional typical domains. Based on their structure, the E2 enzymes can be divided into three groups (Jentsch (1992) Ann. Rev. Genet. 26:179-207)). Class I E2's consist almost exclusively of the conserved domain. Class II proteins have C-terminal extensions that may contribute to substrate recognition or to cellular localization. For example, yeast Ubc2 and Ubc3 have a highly acidic C-terminal domain that promote interaction with basic substrates such as histones (Jentsch (1992) Ann. Rev. Genet. 26:179-207)). Class III enzymes have various N-terminal extensions; however, their function is not known.
Genetic and molecular analysis has revealed that different Ubc's have different cellular functions. Two closely related Ubc's, Ubc4 and Ubc5, appear responsible for ubiquitin-dependent degradation of most short-lived and abnormal proteins (Jentsch (1992) Ann. Rev. Genetics 26:179-207). Ubc2 (RAD6) is required for several functions, including DNA repair, sporulation (Sung et al. (1988) Genes & Dev. 2:1476-1485) and N-end rule degradation (Dohmen et al (1991) Proc. Natl. Acad. Sci. USA 88:7351-7355). Ubc3 (Cdc34) is required for the G1/S transition (Goebl et al. (1988) Science 241:1331-1335), where it appears to participate in the ubiquitin-dependent destruction of the G1 cyclin dependent kinase (cdk) inhibitor, p40.sup.sic1 (Schwob et al (1994) Cell 79:233-244). Ubc9 is required for cell cycle progression in late G2 or early M; both CLB5, an S phase cyclin, and CLB2, an M phase cyclin, are stable in Ubc9 mutants, suggesting that Ubc9 may be responsible for cyclin ubiquitination (Seufert et al (1995) Nature 373:78-81). E2-C, a clam Ubc was determined to be one of the components of the clam oocyte system responsible for the specific ubiquitination of cyclin (Hershko et al. (1994) J. Biol. Chem. 269:4940-4946).
However, heretofore, the Ubc(s) responsible for the ubiquitination of the mitotic cyclins in humans were unidentified and characterized.